In the saccharification industry, pullulanase produced by Klebsiella pneumoniae and the like and isoamylase are known as enzymes which hydrolyze α-1,6-glucopyranoside linkages in starch and amylopectin. Of these, pullulanase catalyzes reversible reactions such as the formation of tetrasaccharides through polymerization of maltose and the transfer of maltose to amylose in the presence of a high concentration of substrates (i.e., 20% (w/v) or more). Therefore, high purity maltose is not predicable when β-amylase is used together in the production of maltose.
Meanwhile, isoamylase is an enzyme which hydrolyzes α-1,6-glucopyranoside linkages in starch, amylopectin, and glycogen, and is known to be capable of producing high purity maltose since it does not catalyze reversible reactions. As isoamylase-producing bacteria, Pseudomonas amyloderamosa, Flavobacterium odoratum (currently called Naxibacter haematophilus), and the like have been reported. However, isoamylases produced by these bacteria are incompatible with various amylases to be used together in terms of the optimum pH and temperature, and the like, and therefore, their capacity could not be fully demonstrated. Generally, the production of starch sugar is carried out at a high temperature of 50° C. or more and under weakly acidic conditions of pH 5.0 to 6.0, which are favorable conditions for other amylases. However, since the optimum pH of isoamylase produced by Pseudomonas amyloderamosa is from 3.0 to 4.0, which is shifted in the acidic region (Non Patent Literature 1), it has been difficult to use this enzyme in combination with malt β-amylase and bacterial β- and α-amylases, which have poor acid resistance. While isoamylase produced by Flavobacterium odoratum (currently called Naxibacter haematophilus) and the like is compatible with various amylases to be used together in terms of the optimum pH, its optimum temperature is as low as 40 to 45° C. (Non Patent Literatures 2 and 3). Therefore, it has been difficult to use this enzyme in combination with various amylases.